APPENDIX A.

OPINION OF PROF. CHRISTIAN KRUPKE
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Prepared by: Christian H. Krupke, Ph.D Associate Professor of Entomology, Purdue 

University, June 7 2012.

A response to a recent review of publications on neonicotinoids and pollinators 

prepared by Heintzelman, Kelly, Fischer and Maus (dated 23 May 2012), 

focusing on work of Krupke et al.(1). Comments of Heintzelman et al., appear in 

italics, response from lead author of the study (C. Krupke) below each comment. 

“Krupke et al. (2012) report the findings of a study on potential routes of 

exposures for honeybees to pesticides, especially to neonicotinoids, conducted 

in a corn growing region in USA in 2010. The study was initiated in response to 

reports of bee kills at Indiana apiaries in spring of 2010 which coincided with the 

corn planting period in the area and which were believed to be related to 

neonicotinoid seed treatment products”.

The study was conducted in both 2010 and 2011 – there were reports of bee kills 

both years. There are currently a number of similar reports in 2012 from Indiana, 

Minnesota, Ohio, and the provinces of Ontario and Quebec in Canada. The 

investigations into the causes of these bee kills are ongoing. 

All dying bees exhibited the convulsions, uncoordinated movements and tremors 

that are typical of neurotoxic insecticide exposure. An analysis of the bees using 

LC-MS/MS revealed that the neonicotinoid clothianidin was present in each case 

(range of 3.8 – 13.3 ppb); these were the only insecticides found in those 

samples. Note that thiamethoxam is rapidly metabolized in insects to clothianidin; 

so it is not possible to identify which of these compounds honey bees were 

initially exposed to. Apparently healthy bees collected from the same apiary 

contained no traces of any insecticides. These data suggest that there is a strong 

likelihood that the neonicotinoids thiamethoxam and/or clothianidin were 

responsible for the bee kills we investigated. 

Corn and soybean seed treatments are the only major use of clothianidin and 

thiamethoxam in our study area. All reports of bee kills occurred when corn was 

being planted nearby. Therefore, the remainder of our study focused upon 

itemizing the potential routes of honeybee exposure to these compounds and 

quantifying the concentrations of a range of pesticides in each potential exposure 

route. 

“This study does not provide any fundamentally new evidence about honeybee 

exposure to neonicotinoid seed treatment products. The exposure levels reported 

in soil, pollen and nectar are generally consistent with previous research and 

were not high enough to represent a significant risk for honey bees.”

While anecdotal reports have persisted for years, there was no documentation 

prior to our study (including, but not limited to, peer-reviewed publications) of the 

potential causes of bee deaths in association with the planting of neonicotinoid 

treated seeds in North America. 
2

Our study took the approach of quantifying pesticide levels in a variety of 

materials that honey bees may be exposed to near plantings of treated seed. We 

found seed treatment insecticides at a wide range of concentrations in virtually 

every sample type we investigated (field soils, dandelions near fields, beecollected pollen stored in cells and collected from pollen traps, maize pollen, and 

exhaust talc from planters). The only sample type that showed no trace of 

neonicotinoids was nectar collected from within the hive. 

The concentrations found in soil samples varied (2.1-9.6 ppb), and these results 

reflect the persistence of these compounds in field soils (2); we found 

neonicotinoids 2 full growing seasons after the treated seed was planted. 

The spurious claim that the concentrations of neonicotinoids we reported in 

stored pollen (2.9-10.7 ppb clothianidin and 6.2-20.4 ppb thiamethoxam, found 

co-occurring) were “not high enough to represent a significant risk for honey 

bees” is not supported by data. Sublethal effects of these compounds remain an 

active area of research and what constitutes a “significant risk” is far from welldefined. Using figures from our study provide a case in point: a bee fed pollen 

containing 20.4 ppb of thiamethoxam for the 10 d period it spends as a nurse bee 

would be exposed to an equivalent of ca. 50% of the oral LD50 of 2.8 ng/bee (3). 

While the effects of such levels of dietary exposure level on individual bees (and 

colonies) remain unclear, dismissing these results out of hand and without 

supporting documentation reflects a gross oversimplification of a complex 

problem. 

“Higher concentrations found in waste talc collected from inside pneumatic 

equipment post-planting represent an intrinsic hazard to honey bees; however 

actual exposure of bees to this material was not demonstrated and would appear 

to be preventable.”

The concentrations found in waste talc were extremely high (up to 15,030 ppm

clothianidin, up to 13,240 ppm thiamethoxam). These concentrations reflect that 

the treatments applied to the seed are partially removed by and become part of 

the used talc/seed debris that constitutes planter exhaust material. The material 

we removed from the planter for our analysis is the same material that ultimately 

enters the environment. Most of this material is exhausted while planting: either 

with the seed and into the seed furrow or into the air behind and above the 

planter via exhaust fans. The remaining material in planters is removed during 

routine cleaning operations. Thus, this material represents a hazard to honey 

bees and other non-target insects that encounter it, because all of it (by design) 

leaves the planter at some point. 

While neonicotinoids were found on dead bees, stored pollen, dandelions etc., 

our methods did not allow us to determine how the materials came to be there. 

However, given the timing and location of bee kills (near corn fields during corn 
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planting) and the extremely high toxicity and potential mobility of waste talc, our 

findings lead us to conclude that this planter exhaust has a role in distributing 

seed treatment pesticides to areas where foraging bees will encounter them. The 

findings of Tapparo et al. (4), published shortly after our work, provides direct 

support for this hypothesis. This research group manipulated honey bee forager 

flight paths so that they flew behind corn planters sowing neonicotinoid treated 

seeds. The foragers returning to the hive were found to be coated with 

insecticides found in the seed treatments, significantly above the published lethal 

levels of 22-44 ng/bee (5,6). 

Clearly, this exposure route may be preventable. However, the current situation

is that there are no recommended methods for minimizing or preventing 

exposure of honey bees to planter exhaust. No recommendations exist for 

disposal of used talc. In short, growers planting treated seeds using currently 

recommended practices for planting and cleaning of equipment will result in large 

volumes of exhaust talc being distributed to the outside environment. 

“In their field experiment, low exposure levels and no adverse effects were 

observed for bee colonies placed “in harm’s way” around the perimeter of a field 

as it was planted with treated corn seeds. Overall, the publication represents an 

interesting case study, but it does not provide any significant new insights into 

exposure of honeybees to neonicotinoid insecticides.”

The first part of this statement is misleading, as it implies that we designed and 

conducted this field study to document adverse effects of planting upon colonies. 

This was never an intended part of the study. There was no measure of colony 

health at any time (before, during or after the study). 

Simply put, we used bee colonies placed near planted fields as pollen collectors. 

We placed pollen traps in each hive and analyzed the bee-collected pollen to 

determine 1) what proportion of pollen collected by foraging bees was corn 

pollen, and 2) whether pollen collected by honey bees in these environments was 

contaminated with pesticides, and whether that level of contamination increased 

following planting of treated seed and associated planter exhaust. 

This portion of our study demonstrated that foraging bees collected large 

amounts of corn pollen while it was present (experiment-wide mean of ca. 44%), 

and that neonicotinoids were found in 10 of 20 samples analyzed (ranging from 

1.2-7.4 ppb thiamethoxam and from 1.1-88 ppb clothianidin). These levels of 

neonicotinoids are up to 10-fold higher than those reported by previous studies of 

bees foraging near treated crops (4). 

Our study and others have shown that seed treatment pesticides are found in the 

pollen of corn and other plants grown from treated seeds (7,8). Corn pollen is 

abundant across large areas of the continental US during late June and early 

July each year, so it is important to determine whether bees use this food 
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resource. The collection of this pollen by foraging honey bees reflects that dietary 

exposure to pesticides occurs later in summer as well, and not solely during the 

spring planting period. 

These linkages between honey bees and seed treatment insecticides were not 

known to our fellow researchers and, more importantly, by the agricultural 

stakeholders who either use seed treatment insecticides or live and work in areas 

where they are used. The response from growers and beekeepers alike has 

overwhelmingly demonstrated that they feel this work identifies critical, previously 

unexplored links between planting of treated seed and honey bees. 

The primary contribution of our studies is two-fold: 1) we document that bee-kills 

were associated with the presence of seed treatment insecticides in each sample 

tested; and 2) evaluate and provide an initial “ranking” (using concentrations of 

neonicotinoids as a guideline) for several honey bee exposure routes. These 

types of data are a key step in determining when and where risks to pollinators 

occur and can be readily mitigated. For example, the extremely high levels of 

seed treatment pesticides found in talc exhausted from planters stands out as a 

potential source of acute exposure and a previously unidentified target for 

remedial action. The lower concentrations found in many other sample types are 

certainly worthy of further study as well, and may act as important routes for 

sublethal exposure. 

References

1. Krupke, C. et al. (2012). Multiple Routes of Pesticide Exposure for Honey Bees Living near 

Agricultural Fields, PloS ONE vol.7.

2. US Environmental Protection Agency, Office of Pesticide Programs. Factsheet

Clothianidin (2003) EPA Publication 7501C; www.epa.gov/opprd001/

factsheets/clothianidin.pdf.

3. Laurino D. et al. (2011). Toxicity of neonicotinoid insecticides to honey bees: laboratory tests. 

Bull Insectol 64: 107–113.

4. Andrea Tapparo et.al. Assessment of the Environmental Exposure of Honeybees to Particulate 

Matter Containing Neonicotinoid Insecticides Coming from Corn Coated Seeds, Environ. Sci. 

Technol., vol. 46, 2592- 2599, 2012;

5. US Environmental Protection Agency, Office of Pesticide Programs. Factsheet Clothianidin 

(2003) EPA Publication 7501C; www.epa.gov/opprd001/factsheets/clothianidin.pdf.

6. Iwasa T. et al. (2004). Mechanism for the differential toxicity of neonicotinoid insecticides in the 

honey bee, Apis mellifera. Crop Prot 23: 371–378.

7. Cutler G. and Scott-Dupree C. (2007) Exposure to clothianidin seed-treated canola has no 

long-term impact on honey bees. J Econ Entomol 100: 765–772.

8. Bonmatin JM et al. (2005). Quantification of imidacloprid update in maize crops. J Agri Food 

Chem 53: 5336–5341.